US2023340073A1PendingUtilityA1
Recombinant factor viii variants

Assignee: CODEXIS INCPriority: Jan 7, 2022Filed: Jan 5, 2023Published: Oct 26, 2023
Est. expiryJan 7, 2042(~15.5 yrs left)· nominal 20-yr term from priority
Inventors:Jeremiah Dallas Heredia Nikki D. Kruse Aksiniya Lyubenova Petkova Marcus Rohovie Adam P. Silverman
C07K 14/755C12N 15/63A61P 7/04A61K 38/00
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References
Claims
Abstract

The present disclosure provides recombinant factor VIII polypeptides, polynucleotides encoding the recombinant factor VIII polypeptides, and compositions thereof, wherein the recombinant factor VIII polypeptides have been engineered to provide improved expression, activity/potency, clotting activity/potency, serum/plasma stability, and/or reduced immunogenicity. The present disclosure also relates to the use of the compositions comprising the recombinant factor VIII polypeptides or polynucleotides encoding the recombinant factor VIII polypeptides for therapeutic purposes.
Claims

exact text as granted — not AI-modified
1 . A recombinant factor VIII polypeptide, or a biologically active fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or to a reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072. 
     
     
         2 . The recombinant factor VIII polypeptide of  claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. 
     
     
         3 . The recombinant factor VIII polypeptide of  claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. 
     
     
         4 . The recombinant factor VIII polypeptide of  claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2360-2870, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs. 4-2346 and 2350-2870, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2. 
     
     
         5 . The recombinant factor VIII polypeptide of  claim 1 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 1186, 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442, or 1452, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2. 
     
     
         6 . The recombinant factor VIII polypeptide of  claim 1 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/K/L/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534F/I/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S/Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/TN/VW, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, 1442T, or 1452L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2. 
     
     
         7 - 9 . (canceled) 
     
     
         10 . The recombinant factor VIII polypeptide of  claim 1 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1148, 85/708/1148, 798, 706, 171, 335, 63, 344, 428, 879, 59, 429, 205, 1264, 797, 1248, 873, 792, 708, 57, 96, 362, 1292, 346, 433, 65, 795, 684/798, 353, 332, 1259, 356, 436, 1245, 1145, 716, 56, 773, 734, 358, 729, 665, 359, 61, 1250, 726, 37/96/171/429/708/799/1148, 171/429/732/1148, 96/171, 171/919/1151, 171/792/797/799/1148, 171/569/1148, 96/171/429/792/797/799, 171/429/708/1422, 171/429, 321/362/732/797/799, 171/1100, 708/1148/1186, 429/724/732/1148, 171/362/795/797/799, 171/429/732, 96/171/643/797/799, 171/792/797/799, 503, 171/732, 1148/1193, 96/171/362/429/732/795/797, 334, 732, 351, 1377, 999, 306, 349, 257, 646, 435, or 58, wherein the positions are relative to the reference sequence corresponding to SEQ ID NO: 2. 
     
     
         11 - 13 . (canceled) 
     
     
         14 . The recombinant factor VIII polypeptide of  claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072, or relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072. 
     
     
         15 . The recombinant factor VIII polypeptide of  claim 14 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution at amino acid position 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 37, 56, 57, 58, 59, 61, 63, 64, 65, 85, 96, 104, 110, 119, 127, 132, 158, 160, 164, 167, 168, 171, 175, 178, 186, 187, 205, 210, 211, 212, 215, 225, 246, 257, 260, 265, 281, 292, 294, 301, 303, 304, 306, 308, 310, 311, 312, 318, 320, 321, 325, 328, 332, 334, 335, 344, 346, 347, 349, 351, 353, 354, 355, 356, 358, 359, 362, 370, 387, 388, 390, 392, 393, 397, 407, 428, 429, 433, 435, 436, 481, 487, 488, 489, 491, 498, 499, 503, 514, 527, 533, 534, 535, 536, 538, 543, 550, 551, 555, 558, 569, 580, 581, 584, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 643, 646, 650, 665, 667, 676, 680, 681, 682, 683, 684, 686, 693, 696, 698, 700, 706, 707, 708, 712, 716, 724, 726, 729, 731, 732, 734, 735, 736, 737, 738, 739, 740, 744, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 759, 770, 773, 775, 785, 786, 787, 790, 791, 792, 793, 794, 795, 797, 798, 799, 800, 801, 802, 805, 806, 807, 808, 809, 810, 811, 812, 815, 816, 817, 818, 819, 821, 822, 824, 825, 826, 828, 830, 831, 836, 837, 838, 839, 840, 841, 844, 866, 873, 879, 892, 896, 897, 903, 907, 914, 916, 917, 918, 919, 921, 922, 923, 924, 926, 929, 938, 939, 945, 948, 951, 968, 999, 1037, 1040, 1041, 1042, 1043, 1049, 1050, 1051, 1053, 1054, 1076, 1078, 1080, 1084, 1088, 1089, 1092, 1093, 1094, 1095, 1098, 1099, 1100, 1101, 1104, 1108, 1109, 1113, 1114, 1116, 1127, 1129, 1138, 1145, 1148, 1151, 1156, 1158, 1160, 1186, 1192, 1193, 1194, 1205, 1210, 1218, 1220, 1221, 1222, 1224, 1229, 1245, 1248, 1250, 1259, 1264, 1269, 1270, 1274, 1281, 1283, 1292, 1297, 1339, 1352, 1373, 1377, 1378, 1380, 1389, 1414, 1422, 1427, 1428, 1439, 1440, 1442, or 1452, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072. 
     
     
         16 . The recombinant factor VIII polypeptide of  claim 14 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or amino acid residue 13G/Y, 14G/M/V, 15G/S, 16A/D/G/P/V, 17S/V, 18L/R, 19G, 20L, 21M, 24V, 37H, 56E/P, 57A/L/S/T/V, 58K, 59D/G/L/M/Q/R/S/V/W, 61Q/S, 63C/E/F/G/H/I/K/L/M/N/Q/R/S/T/W/Y, 64D/G, 65A, 85Q, 96I/L/R/V/W, 104S, 110H, 119G/V, 127V, 132P/R, 158G, 160E/T, 164E, 167G/H/R, 168G/R, 171A/D/E/F/H/K/N/M/P/Q/R/S/T/V/W/Y, 175A/G/H, 178C/M/Q/V/W, 186A, 187C/G, 205R, 210P, 211G/K/P/Q/R, 212A/F/G/L/K/M/N/P/R/S/V/Y, 215E/P/S, 225R, 246D, 257I, 260E/K/T, 265R/S/W, 281A/S, 292D, 294G, 301K/M, 303G/T, 304A/E/G, 306A/D/G/P/S/T, 308P, 310G/V, 311E/G, 312L, 318C/G/K/L/M/Q/R/T/V, 320G/I/L/P/Q/R/S/T/V, 321Y, 325Y, 328G/Q/S, 332A/F/G/H/N/P/Q/R/T/V/W/Y, 334G, 335E/P, 344A/E/L, 346R/V, 347N, 349G, 351S, 353S, 354R, 355C, 356L/N, 358K/S, 359E/G, 362E/G/N/P/T/V/W, 370A, 387A/G/I/L/Q/R/S/T/V/W, 388A/P, 390A/E/G/L/M/P/S/T/V, 392A, 393A/F/G/H/M/Q/R/W/Y, 397G/N, 407V, 428F, 429L/S, 433A/G, 435Q, 436D/G, 481V, 487N, 488G, 489E/F/Q/R, 491E, 498A, 499C/L, 503S, 514L, 527A, 533R/V/Y, 534I/F/L/M, 535C/G/N, 536A/G/K/L/M/P/R/W, 538V, 543G/L/P/W, 550G, 551W, 555E/G/R/T, 558A/E/G/L/P/Q/R/S/T/V/W, 569T, 580S1Y, 581K/V, 584A, 610H/K/L/R/S/T, 611D/E, 612G/S/T, 613S, 614A/F/H/I/N/T, 615D/N, 616D/E/G/Q/W, 617D/E/G/N, 618C/E/G/P/Y, 619E, 620N/P, 621K/M/R/T/W, 622D, 643N, 646I, 650M, 665G, 667I, 676Q, 680L/R, 681A/C/E/F/G/L/N/R/Q/S/T/W, 682R, 683W, 684G/K, 686W, 693T, 696E, 698D/S/W/Y, 700A/E/Q/T/V, 706A/G/M/Y, 707H, 708A/K/T/V, 712G/L/M/Q/S/T/Y, 716K/L/W, 724I, 726A/L, 729T, 731I/L/R, 732C/D/E/G/L/R/W/K/M/Q/S/T/V/Y, 734I/R/S, 735D/S/V, 736F/G, 737F/G/K/L/M/N/S/V, 738A/D/E/F/H/K/N/P/R/S/T/Y, 739G/P/R, 740G, 744A/R, 746P, 747D, 748F/G/K/N/S/V, 749A/C/D/F/G/I/K/L/M/P/Q/R/S/T/V/Y, 750A/D/G/K/M/N/P/Q/R/S/T/V/W, 751A/D/E/Y, 752D/E/F/G/L/M/Q/R/S/V, 753D, 754W, 755G/L/P/Q/S/Y, 756G/P/R/T, 757A/G/K/T, 759S, 770W, 773H, 775E/G/K/N/P/S/V, 785V, 786C/D/L, 787W, 790A/G/I/M/P/W, 791M/T, 792H/W/Y, 793G, 794R, 795C/N/S/T, 797C/G/P/Q/T/W/Y, 798C/R/T, 799A/E/G/M/P/Q/R/S/V, 800C/G/I/L/M/N/P/Q/R/T/V/W/Y, 801A/G/I/R, 802S/V, 805C/G/H/K/L/M/P/Q/R/S/V/W, 806E/G/R/S, 807C/L/P/R, 808K/S, 809A/C/G/I/K/L/N/Q/R/S/T/V/W, 810V, 811F, 812G/I/V, 815I, 816V/W, 817A/D, 818A/E/G/P/S/V/W, 819V, 821Q, 822S/T, 824W, 825L, 826G, 828K, 830M/V, 831W, 836G/R/V/W, 837G, 838G/T, 839A/G/K/P, 840G, 841W, 844H, 866A, 873L/Q/T, 879Q, 892T, 896L/V, 897G/K/R, 903E, 907D, 914V, 916C/E/I/K/L/M/Q/T, 917G, 918M/S, 919G, 921R/W, 922W, 923M, 924I/M/P/Q/W, 926T/V, 929A/Q/V, 938R/V, 939E/S, 945K, 948S, 951V, 968A/P, 999R, 1037H/M/N/P, 1040E, 1041G/H/N, 1042A/E/T, 1043E/R, 1049G/W, 1050E/P/Q/S/T, 1051E/H/K/R/S/W, 1053A/R, 1054C, 1076G/S, 1078S, 1080G/V, 1084L/N, 1088G/S, 1089F/S, 1092G, 1093A/E/G/M/N/P/Q/R/S/T/V/W/Y, 1094S, 1095G, 1098G, 1099G, 1100I/S, 1101G, 1104C/E, 1108M/S/V/W, 1109G, 1113F, 1114A/M, 1116A/G/I/L/R, 1127L/V, 1129P, 1138Q, 1145R, 1148A/C/E/L/M/N/Q/R/S, 1151S, 1156D/G/N/R, 1158N, 1160R, 1186E, 1192D, 1193A/E/G/I/L, 1194P, 1205L, 1210M/T/V/W, 1218A/G/L/M/P, 1220D/E/L/T, 1221G/S/T/V, 1222F, 1224P, 1229D, 1245R, 1248A/C/D/E/F/G/H/I/K/M/P/Q/R/S/T/W/Y, 1250A/P/S/T, 1259T, 1264L/R/W, 1269F, 1270V, 1274N/V, 1281H/K/P/Q/R/S, 1283E/H/L/S/T, 1292Y, 1297A, 1339C, 1352E, 1373S, 1377A/C/D/E/G/H/I/M/P/R/S/V/T/Y, 1378A/C/D/E, 1380A/G/W, 1389W, 1414A/C/D/E/G/H/N/P/T/W, 1422I, 1427Y, 1428G, 1439A/E/H/L/S/T, 1440A/C/E/G/S/T, or 1442T, 1452L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252, 504, 706, 840, 1200, 1514, or 2072. 
     
     
         17 - 19 . (canceled) 
     
     
         20 . The recombinant factor VIII polypeptide of  claim 14 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252. 
     
     
         21 . The recombinant factor VIII polypeptide of  claim 20 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1380, 388, 1043, 610, 1274, 1220, 1442, 1193, 621, 1054, 755, 1041, 748, 1049, 1221, 1051, 612, 1037, 616, 387, 489, 750, 1224, 618, 1050, 1053, 896, 897, 749, 260, 481, 613, 756, 63, 265, 752, 1218, 1378, 614, 1440, 1439, 1205, 1222, 1389, 1269, 390, 397/1218, 757, 1373, 346, 63/1248, 349, 306/355/1422, 1422, 306/346/708, 1148, 171/346/1248, 63/797/799/1248/1422, 171/799, 362, 1248, 171/306/346/999/1248, 171/1148, 734/799/1248/1422, 63/732, 63/171/306/346/732, 171, 171/1422, 63/362/732/799/1248, 999/1148/1422, 346/349, 306/349, 63/171/1148/1151, 306, 344/349/354/1250, 1250, 63/306/344/349/354/503/1248, 59/63/332/349/503/706/708, 63/306/344/349/354/706/1248, 59/63, 332/706/708/1248/1250, 503, 59/63/349/354, 59/349/708, 59/306/344/1248, 344/1248/1250, 708, 63/344/349, 59, 63/344/349/354, 706, 59/306/770, 63/347/354/1248, or 354/503, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 252. 
     
     
         22 . (canceled) 
     
     
         23 . The recombinant factor VIII polypeptide of  claim 14 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or to the reference sequence corresponding to SEQ ID NO: 504, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 504, or relative to the reference sequence corresponding to SEQ ID NO: 504. 
     
     
         24 . The recombinant factor VIII polypeptide of  claim 23 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 750, 1051, 260, 756, 1193, 332, 897, 387, 1037, 306/749, 1270, 489, 487, 488, 903, 171/1248/1440, 260/1148/1378, 171/260/387/750/1248, 171/260, 171/260/387/750/1051/1148, 171, 750/1051/1148, 171/260/756, 171/1248, 171/1148, 59, 1440, 332/750/1248, 756/1248, 1248, 171/387/1248/1440, 750/1440, 59/171/750, 171/1193/1248, 750/1148/1193/1440, 1148, 171/387/1051, 748, 306, 306/387/390/749/1220, 390, 306/390/1037, 306/612/749, 387/390/749, 63/390/503/749, 1220, 63/306, 749, 306/390/749/1037, 306/749/1220, 306/387/390/612/749/1037, or 328, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 504. 
     
     
         25 . (canceled) 
     
     
         26 . The recombinant factor VIII polypeptide of  claim 14 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or to the reference sequence corresponding to SEQ ID NO: 706, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 706, or relative to the reference sequence corresponding to SEQ ID NO: 706. 
     
     
         27 . The recombinant factor VIII polypeptide of  claim 26 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1051, 1221, 1218, 387, 749/1218, 332, 897, 696, 816, 824, 826, 393, 825, 698, 332/387/390/1148, 328/387/749, 749, 306/328/332/390/1051/1193/1218, 63/306/332/1051/1148, 63/306/332/387/1051, 387/390/1051, 186/306/328/332/390/749/1051/1148/1193, 387/390/756/1051, 306/390/1051, 306/332/390/749, 306/387/390/1148, 306/328/332/387/390/749/1051/1148/1193, 332/387/390/749/1148, 387/390/749, 306/387/749/1051/1148, 387/749, 306/945/1051/1193/1221, 306/390/749/1051, 63/306/332/390, 306/387/390/749/1148/1193/1221, 306/332/387/390/749/1218, 63/306/332/390/1051/1148/1193/1218, 924, 755, 752, 751, 923, 797, 754, 793, 753, 922, 896, 938, 775, 732, 810, 747, 393/407, 819, 787, 802, 811, 392, or 397, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 706. 
     
     
         28 . (canceled) 
     
     
         29 . The recombinant factor VIII polypeptide of  claim 14 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 840, or to the reference sequence corresponding to SEQ ID NO: 840, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 840, or relative to the reference sequence corresponding to SEQ ID NO: 840. 
     
     
         30 . The recombinant factor VIII polypeptide of  claim 29 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 620, 491, 1229, 615, 1283, 822, 328, 212, 818, 555/684, 828, 550, 830, 839, 13, 14, 17, 836, 1210, 1281, 64, 682, 318, 20, 21, 325, 533, 19, 817, 831, 225, 1156, 393/747, 753, 393/732/751, 63/797, 622, 617, 907, 1040, 611, 488, 1192, 1194, 619, 16, 802, 211, 555, 536, 527, 841, 320, 837, 558, 914, 312, 681, 838, 15, 1427, 18, 683, 24, 534, 840, 1160, 551, 393, 393/747/752/811/819/923/1148, 393/751/775, 63/393/732/775/1148, 393/732/1148, 775/1148, 393/752/753/775/793/797, 393/923, 393/923/1148, 393/747/751, 393/747/753/775/1148, 752/923/1051, 63/775, 63/393/752/775/793/1051/1053, 752/753/775/793/819, 63/1051, 63/747/752/753/775/811/819/923/1051, 393/752/787, 751, 63/1148, 63/747/752/753/819/923/1148, 1148, 747/752/753/793/811, 63, 797/1148, 751/752/753/1051, 63/732/1051, 752/797/815/923/1051/1148, or 747/753, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 840. 
     
     
         31 . (canceled) 
     
     
         32 . The recombinant factor VIII polypeptide of  claim 14 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1200, or to the reference sequence corresponding to SEQ ID NO: 1200, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1200, or relative to the reference sequence corresponding to SEQ ID NO: 1200. 
     
     
         33 . The recombinant factor VIII polypeptide of  claim 32 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 681, 622, 617, 558, 536, 211, 320, 839, 797, 533, 751, 303, 951, 164, 818, 807, 799, 735, 794, 926, 755, 921, 801, 1094, 968, 929, 924, 698, 168, 581, 916, 1428, 805, 1114, 311, 737, 693, 809, 757, 1099, 676, 740, 791, 211/536/622/681, 536/558, 211/536/555/617/732, 211/681/732, 211/536/555, 555, 211/536, 211/212, 211/536/617/681/732, 211/536/558/617/681, 555/732, 536/555/558/617/732, 536/617/681/732, 536/555, 536/622/681, 732, 212/320/533/617/681, 211/312/533/534/617/619/681/839, 212, 211/212/320/619/681/839, 797/839, 212/312/320/533/534/617/681, 558/619/681, 320/558/681, 212/320/534/619/681/797/839, 320/558/619/732/839, 211/212/312/534/558/619/681/732/839, 320/534/558/619/732/797, 558/617/619/681, 211/534/558/611/681, 211/320/617/619/681, 558/617/681/797, 320/534/619/732, 211/619/681, 211/212/534/558/619/732/839, 212/320/619/681/797/839, 212/320/611/839, 320/558/619/681, 312/533/617/681/797/839, 558/611/681, 312, 211/320/558/619/681/732/797/839, 320/558/732/797, 533/558/611/619/716/732/839, 312/681, 320/534/619/681, 211/320/533/536/619, 558/617/681/839, 212/320/534/558/681, 533/534/681/839, 212/534/619/681, 617/619/681, 211/212/320/558/617/681, 212/534/558/681/797, 212/320/534, 211/312/619, 212/312/619/839, 210/212/320/558/681, 212/312/534/681/797/839, 211/320/558/839, 294, 759, 538, or 1109, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1200. 
     
     
         34 . (canceled) 
     
     
         35 . The recombinant factor VIII polypeptide of  claim 14 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 1514, or to the reference sequence corresponding to SEQ ID NO: 1514, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 1514, or relative to the reference sequence corresponding to SEQ ID NO: 1514. 
     
     
         36 . The recombinant factor VIII polypeptide of  claim 35 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises at least a substitution or substitution set at amino acid position(s) 1042, 1041, 1051, 1283, 1281, 916, 187, 215, 178, 1050, 1210, 104, 615, 610, 536, 618, 621, 614, 110, 301, 1078, 812, 786, 127, 175, 1093, 1088, 650, 535, 806, 738, 700, 132, 1076, 1084, 498, 756, 1108, 746, 160, 543, 310, 584, 918, 1452, 790, 292, 800, 167, 892, 304, 1116, 752, 281, 917, 712, 1080, 806/1352, 1095, 744, 1098, 948, 119, 785, 1113, 667, 158, 580, 707, 731, 1100, 1089, 1092, 939, 1104, 736, 1297, 1127, 686, 1129, 1138, 786/821, 680, 1101, 808, 739, 308, 536/555/619/732/799/805, 555/619/801, 555/619/799, 619/799/801/805, 536/555/619/732/799, 799/805, 801, 246/619/799/801, 536/555/619, 619/839, 619/799/805, 536/801/805, 555/619/799/805, 619/801/805, 555/619, 536/619/801, 801/805, 536/619/799/805, 555/619/732/799/801/805, 749, 616, 611, 370/614, 1156, 1158, 555/681/737/799, 536/619/809/1109, 536/555/619/737/799/809/839/916/921, 916/1109, 536/619/681/799/916/1109, 555/619/681/807/1109, 555/619/681/737/799, 619/681/799/916/1109, 619/681, 555/921, 619/681/916, 555/619/681/799, 555, 732, 619/799, 619, 681, 555/916, 839, or 799, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1514. 
     
     
         37 . (canceled) 
     
     
         38 . The recombinant factor VIII polypeptide of  claim 14 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence corresponding to residues 20-1457 of SEQ ID NO: 2072, or to the reference sequence corresponding to SEQ ID NO: 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-1457 of SEQ ID NOs: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072. 
     
     
         39 . The recombinant factor VIII polypeptide of  claim 38 , wherein the amino acid sequence of the recombinant factor VIII comprises at least a substitution or substitution set at amino acid position(s) 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167, 167/738/1076/1093, 167/738/752/800/805/808/809/1116, 167/800/809/1076, 167/318, 167/318/800/805/808/809, 167/318/681, 167/318/681/700/805/808/809/1076/1116, 167/318/700, 167/318/700/1076, 167/318/700/738/739/1076, 167/681/738/800/808/809, 167/178/700/752/1093, 167/700, 167/700/738/739/1093, 167/700/738/752/1093, 167/700/738/752/805/808/809/1093/1116, 808/809, 1076, 738, 738/808/809, 738/1076, 738/739, 738/739/809, 738/739/809/1093/1116, 738/739/1093, 738/739/752/1093, 738/739/799/800/1093, 738/739/1116, 738/800, 738/1093, 738/752, 738/752/1093, 738/752/799, 738/799/809/1093, 738/799/1093, 739, 739/800/809, 800, 800/805/809, 809, 809/1093, 809/1093/1116, 809/1116, 318, 318/1076, 318/1076/1116, 318/739/805/808/809/1076, 318/499/700/738/809, 318/1093, 318/752/1093, 318/1116, 318/543/739, 318/543/681/752/1116, 318/543/1093, 318/700/738/1093/1414, 681, 681/738, 681/738/739/1093, 681/738/739/752/799/800/809/1116/1339, 681/738/739/1116, 681/738/800, 681/800/805/809, 681/809/1093, 681/752/1093, 681/752/1093, 681/700, 681/700/738/739/1093/1116, 681/700/809, 681/700/1093, 681/700/752/808/809/1093/1116, 681/700/752/1093, 681/700/844, 681/700/805/809, 1093, 1093/1116, 752, 752/1076, 752/800/809, 752/800/1093, 752/809, 752/866, 752/1093, 752/1116, 178/318/739/752/809/1076/1116, 178/318/800/809, 178/318/681/800/808/809/1076, 178/681, 178/681/738/1116, 178/752/809, 178/700/800/805/808/809, 178/805/809/866, 1116, 543, 543/738/739/809, 543/738/809/1093, 543/739/1093, 543/800/1093/1116, 543/809/1093, 543/809/1093/1116, 543/681/738/739/752/809/1093, 543/681/738/739/752/1093, 543/681/739/752/800/805/809/1093, 543/681/700/738/739/800/1093/1116, 543/681/700/738/739/805/808/809, 543/681/700/752, 543/681/700/752/809/1093, 543/1093, 543/700, 543/700/738/739/752/800/1093/1116, 543/700/809, 543/700/1093, 543/700/752, 543/700/752/800/1093, 543/700/752/809/1116, 543/700/752/1093, 700, 700/738/739/800/1093, 700/738/739/799/1093, 700/738/800/1093, 700/738/1116, 700/809, 700/809/1093, 700/1093, 700/1093/1116, 700/752, 700/752/808/809/1093/1116, 700/752/809/1093, 700/752/799/1116, 700/752/805/809/1093, 700/799/800, 700/1116, 700/805/808/809/1076, 514/543/681/805/808/809, 805/808/809, or 805/809/1116, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 2072. 
     
     
         40 - 42 . (canceled) 
     
     
         43 . The recombinant factor VIII polypeptide of  claim 1 , wherein the amino acid sequence of the recombinant factor VIII polypeptide comprises residues 20-1457 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-2346 and 2360-2870, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 4-2346 and 2350-2870, wherein the amino acid sequence optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. 
     
     
         44 - 49 . (canceled) 
     
     
         50 . The recombinant factor VIII polypeptide of  claim 1 , wherein the recombinant factor VIII polypeptide exhibits at least one improved property selected from: i) increased activity/potency; ii) increased clotting activity/potency; iii) increased stability to plasma or serum; iv) increased thermostability; v) reduced immunogenicity; vi) increased expression and/or secretion from cells that are transfected with an expression vector; vii) increased transduction and/or secretion from cells that are transduced with a gene therapy vector, or a combination of any of i), ii), iii), iv), v), vi, and/or vii), as compared to a reference factor VIII polypeptide having a sequence corresponding to SEQ ID NO: 2, 252, 504, 706, 840, 1200, 1514, or 2072. 
     
     
         51 . (canceled) 
     
     
         52 . The recombinant factor VIII polypeptide of  claim 1 , comprising a deimmunized recombinant factor VIII polypeptide. 
     
     
         53 . (canceled) 
     
     
         54 . The recombinant factor VIII polypeptide of  claim 1 , wherein the recombinant factor VIII polypeptide is purified. 
     
     
         55 - 58 . (canceled) 
     
     
         59 . A recombinant polynucleotide encoding at least one recombinant factor VIII polypeptide set forth in  claim 1 . 
     
     
         60 - 64 . (canceled) 
     
     
         65 . An expression vector comprising the recombinant polynucleotide of  claim 1 . 
     
     
         66 . (canceled) 
     
     
         67 . (canceled) 
     
     
         68 . A host cell comprising the expression vector of  claim 1 . 
     
     
         69 . (canceled) 
     
     
         70 . (canceled) 
     
     
         71 . A method of producing a factor VIII polypeptide, comprising culturing the host cell of  claim 68 , under conditions such that the factor VIII polypeptide encoded by the recombinant polynucleotide is produced. 
     
     
         72 . (canceled) 
     
     
         73 . (canceled) 
     
     
         74 . A pharmaceutical composition comprising the recombinant factor VIII polypeptide of  claim 1 . 
     
     
         75 . The pharmaceutical composition of  claim 74 , further comprising a pharmaceutically acceptable carrier and/or excipient. 
     
     
         76 . (canceled) 
     
     
         77 . A method for treating and/or preventing the symptoms of deficiency in factor VIII in a subject, comprising administering to a subject having a deficiency in factor VIII an effective amount of the recombinant factor VIII polypeptide of  claim 1 . 
     
     
         78 . The method of  claim 77 , wherein the subject has hemophilia A. 
     
     
         79 - 82 . (canceled)
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